In this translational research proposal we test the hypothesis that healing of chronic cutaneous wounds may be enhanced by grafting of temporary human skin substitutes generated from human keratinocytes which have been genetically engineered to enhance vascularization of the underlying wound bed. Two strategies to achieve this goal are described, I.) tissue-specific expression of the angiogenic growth factor VEGF-165 and II.) tissue-specific expression of a constitutively active transcription, factor hypoxia-inducible factor-1alpha (HIF-1a). HIF-1a is a universal regulator of cellular and systemic oxygen homeostasis and is overexpressed during wound healing. HIF is known to upregulate the expression of a number of target genes including those involved in vasculogenesis. We generated genetically engineered human keratinocyte cell lines ex vivo that constitutively produce VEGF-165 and active HIF-1a protein in a tissue-specific manner regardless of oxygen tension. Using tissue engineering strategies these keratinocyte cell lines will be used to produce biologically active human skin substitutes to investigate the physiological and/or pathological consequences of persistent epidermal angiogenic signaling on vascularization and wound healing. The revised aims are: Specific Aim 1. Isolate stable NIKS keratinocyte lines (NIKS(Empty Vector) , NIKS(VEGF165), NIKS(HIF-1alpha/ODD), NIKS(HIF-1alpha)).Verify that monolayer cultures of these new NIKS cell lines produce and secrete enhanced levels of biologically active angiogenic proteins. Specific Aim 2. Produce human skin substitutes using genetically engineered NIKS(Empty Vector), NIKS(VEGF-165), NIKS(HIF-1alpha), and NIKS(HIF-1alpha/ODD) keratinocytes and analyze for target gene expression and angiogenic protein production. Analyze the histological features of genetically engineered tissues by immunohistochemistry. Determine HIF-1a target gene expression by in situ hybridization and quantitative PCR. Confirm enhanced levels of biologically active angiogenic proteins using quantitative assays. Specific Aim 3. Determine the effects of constitutive, epidermal angiogenic signaling on wound healing, physiological and pathological vessel production, organization, and function using human skin substitute tissue xenografts generated from NIKS, NIKS(Empty Vector), NlKS(VEGF-165) , NlKS(HIF-1a), and NIKS(HIF-1a/ODD) cell lines.